Biological systems perceive extracellular signals, such as light, smell, nerve-nerve stimuli etc., by the initiation of coupled, cascade-like amplification reactions. In many of these, the initial or intermediate steps in the cascade, involve the opening of membrane-associated ion-channels. In ligand-activated channels, the process is initiated by the binding of the small effector molecule (neurotransmitter, odorant, or flavour) to a specific receptor that is either structurally or functionally coupled to the channel protein. This induces conformational changes in the channel protein that leads to the opening of a pore across the lipid bilayer causing a step increase in the membrane's electrical conductance. Biological channels may be functionally reproduced in artificial lipid bilayers leading to effectorinduced currents essentially similar to those occurring in the biological membranes. By means of the electronic amplification available today, a single channel opening event can be detected.
Small amphiphilic peptides, synthetic or of natural origin, were shown to form ionic channels in artificial bilayers. The conductive path across the bilayer is formed by coordinated aggregation of several peptides to create the walls of an aqueous pore. In addition, by modifying their primary sequences, specific channel properties can be altered. Independently, channel formation can be controlled by restricting the lateral and rotational mobility of the peptides in the plane of the membrane.
An ion channel is therefore a device that controls the flow of ions through the dielectric formed by the core of the lipid bilayer. If a bilayer can be attached to a sensing electrode in such a way that: 1) it is highly stable and 2) it preserves its capacity to serve as a medium in which proteins and peptides may form channels, then a unique type of biosensor may be created.
Interfacing layers of amphipathic molecules with solid surfaces has been known for a long time. The methodology introduced by Langmuir and Blodgett is still used in the development of devices that involve adsorption of phospholipids to electrodes. By this methodology, monolayers of the amphipathic molecules are successively transferred from a water-air interface to a solid surface by its orderly dipping in and pulling out from the water compartment. Due to the amphipathic nature of the molecules and the order of the passes of the solid surface through the water-air interface, the layers alternate their hydrophobic-hydrophilic directionality with respect to the solid surface, thus forming stacked bilayers. When these layers are prepared under humidifying conditions, few water molecules with restricted mobility are trapped within the bilayers. However, these systems cannot support bulk-type solvent water molecules between the bilayers, since a liquid-like interface would actually allow the detachment of outer layers from the inner stacked matrix. Consequently, it is clear that a bulk aqueous medium between the first layer and the electrode surface is absolutely inadmissiable.
The lack of bulk solvent water in all Langmuir-Blodgett devices makes them inadequate for mimicking of biologicallike systems and, in particular, for the incorporation of functional ion-channel-forming polypeptides.
Two examples of devices of the prior art prepared according to the Langmuir-Blodgett methodology are to be found in the Australian Patent Application AU 40123/85 and in the PCT International Application published under No. WO 89/01159.
In AU 40123/85 a solid state electrochemical sensor is disclosed which utilizes a film or membrane adapted to pass ions when selected materials, which are to be detected, are present at the membrane surface. In particular, the electrochemical sensor includes a base substrate, and a layer of material attached to the base substrate for producing electrical current in response to the transport of ions to the layer--this layer actually transforms or converts ionic current to electronic current. Also included is a membrane attached to the layer for transporting ions to the layer from a fluid containing the material or chemical species to be detected, the membrane including gating molecules which interact with the chemical species to thereby allow ions from the fluid to permeate the membrane.
WO 89/01159 describes a membrane comprising a closely packed array of self-assembling amphiphilic molecule, and is characterized in that it incorporates plurality of ion channels, and/or at least a proportion of the self-assembling molecules comprising a receptor molecule conjugated with a supporting entity. The ion channel is selected from the group consisting of peptides capable of forming helices and aggregates thereof, coronands, cryptands, podands and combinations thereof. In the amphiphilic molecules comprising a receptor molecule conjugated with a supporting entity, the receptor molecule has a receptor site and is selected from the group consisting of immunoglobulins, antibodies, antibody fragments, dyes, enzymes and lectins. The supporting entity is selected from the group consisting of a lipid head group, a hydrocarbon chain(s), a crosslinkable molecule and a membrane protein. The supporting entity is attached to the receptor molecules at an end remote from the receptor site. It also discloses a biosensor comprising such a membrane bilayer attached to a solid surface.